Isolation and partial characterization of an indigenous plasmid from the plant- associated bacterium Ochrobactrum sp. 11a

JOFRE, E.; PRíNCIPE, A.; FISHER, S.; PISTORIO, M.; LAGARES, A.

Bariloche

Congreso; International Plasmid Biology Conference 2010; 2010

International Society for Plasmid Biology and other Mobile Genetic Elements

Plasmids play a key role for adaptative traits that might be advantageous for survival under local selective pressures providing an important source of novel functions for biotechnological applications. The plant growth-promoting rhizobacterium, Ochrobactrum sp. 11a -an isolate obtained from the endorhizosphere of maize- displays a high intrinsic tolerance to different environmental stresses being capable of using a variety of substrates for growth like sugars, xenobiotic, and phenolic compounds suggesting sophisticated mechanisms for their survival. Members of á-proteobacterial genus Ochrobactrum have been described to possess varied life-styles, such as nitrogen-fixing bacteria in plant nodules, xenobiotic-degrading bacteria, and opportunistic human pathogens. These contrasting life styles could be related to the exceptional level of genomic diversity between Ochrobactrum species mainly due to the occurrence of large plasmids. However, the role of auxiliary genes carried by these replicons remains unclear. We are interested in the characterization of a plasmid present in Ochrobactrum sp. 11a in order to gain information about the role of this replicon in the bacterial survival under different environmental conditions. Methods. Plasmid profiles were visualized by electrophoresis in horizontal agarose gels through a modification of the method of Eckhardt (1978). The mobilization of p11a, was carried out by bacterial matings as described by Simon et al. (1983) using Ochrobactrum sp. 11a harbouring the plasmid p11a::aaCc1 as donor and A. tumefaciens UBAPF2 as the recipient strain. Plasmid p11a was purified by means of a CsCl-ethidium bromide gradient and used for the construction of a partial library pRK442. One clone carrying a 13-kb fragment from p11a was sequenced (pRK442-23). Searches for homologies were performed at the NCBI database by using the BLASTP algorithm (Altschul et al., 1997). Results. Plasmid profiles of the strain Ochrobactrum sp. 11a revealed the presence of a ca. 180 kb cryptic plasmid. This plasmid, called p11a, was found to be self-transmissible. The generation of a library from p11a and the further sequencing of a 13-kb region allowed us to identify genes with similarity to members of the lipase-hydrolase and major facilitator superfamily which could be associated to the utilization of different substrates. Additionally, one ORF encoding a putative enzyme 4-phytase with a high degree of similarity (67%) to the corresponding protein of Rhodobacter strain SW2 was detected. These enzymes, belonging to the class of phosphatases, are involved in the decomposition of phytate, which is widely regarded as an abundant form of soil organic phosphorus. Indeed, E. coli harbouring the recombinant plasmid pRK442-23 became able to decompose sodium-phytate. This putative phytase on p11a may be responsible for the phosphate-solubillizing phenotype exhibited by Ochrobactrum sp. 11a. Phylogenetic analysis from each ORF showed a high similarity to related proteins from both pathogenic and symbiotic strains belonging to the genus Burkholderia and Mesorhizobium, respectively.